Published: November 6th, 2018
The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).
The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.
Gastrulation and the accompanying morphogenetic movements are crucial for shaping the mouse embryo1. The changes in cellular shape and organization during morphogenesis dictate positional information to regulate cell fate and also allow the ensuing signaling pathways to precisely perform their functions to diversify the newly formed germ layers1. The formation of transient organizing structures and signaling centers such as the node and notochord is essential for the execution of the developmental program2. Developmental biologists have used a variety of techniques to study the morphogenesis of these structures, most notable of which is the use of cellular reporters and live ex vivo imaging to follow the dynamics in cellular and subcellular behavior2,3,4. In this report, we focus on describing the details of our optimized protocols for two of these techniques: scanning electron microscopy (SEM) and whole mount immunofluorescence (WMIF), which were and are still instrumental in studying the morphogenesis of the node and the notochordal plate, the precursor of the notochord.
The mouse embryonic node is a teardrop-shaped cup of cells that is located on the ventral surface of the mouse embryo around the early to late head fold stages during gastrulation and morphogenesis (embryonic day, E7.5-E8)2,5,6,7. The notochordal plate morphologically emanates anteriorly from the node3. Each cell in the node and notochordal plate is characterized by a single cilium that protrudes to the outside, which is longer in node cells but whose length varies with the developmental stage2. The rotation of cilia in the node pit has been shown to be important for signaling that determines left-right asymmetry4. The notochordal plate is the precursor of the notochord, the signaling center that is important for the patterning of the adjacent somites and the overlying neural tube3.
Because of the attributes of location (surface), shape (cup) and possessing distinct outer cellular structures (cilia), SEM has traditionally been used to visualize the node and notochordal plate and study their formation and structure2,7. SEM is also used to study the changes in the structure of the node itself or the cilia on its cells in mutations that affect gastrulation, morphogenesis, as well as cilia formation8,9,10. SEM is a technique that utilizes a focused beam of electrons to interrogate the topological ultrastructure of the outer surface of materials such as biological specimens11. The sample is usually fixed, dried and then sputter-coated with metals for observation under a scanning electron microscope as we describe in Step 1.
WMIF is a staining technique to visualize gene products, such as proteins, in three-dimensions (3D). WMIF of tissues, organs or even whole organisms provides spatial information about the distribution of the signal and the shape of the resulting structure in 3D. The technique is based on fixing the sample then staining it with fluorescent conjugates. Mouse embryos ~ E7.5 are small and transparent and therefore ideal for WMIF protocols to visualize the node and notochordal plate. For example, the transcription factor Barchyury (T) is expressed in the nuclei of the node and notochordal plate, and to a lesser extent in the primitive streak, around E7.5-E8 of embryonic development and good working antibodies against T by WMIF are commercially available and make the staining procedure possible. The cells of the node and notochordal plate are also characterized by constricted apical surfaces, which face the outside and thus can be stained with fluorescence-conjugated Phalloidin to mark F-Actin at the apical constrictions. Using these reagents as examples, the combination of T and F-Actin staining by WMIF provides a representation of the node and notochordal plate in 3D in gastrulating mouse embryos as we demonstrate in Step 2 8. However, markers of cilia, such as ARL13B or acetylated tubulin, as well as other markers of the node and notochordal plate, such as FOXA2, can also be used to perform WMIF on developing mouse embryos3,4.
We have shown that striatin-interacting protein 1 (STRIP1) is essential for normal gastrulation and morphogenesis in the mouse embryo8. STRIP1 is a core component of the striatin-interacting phosphatases and kinases complexes (STRIPAK), which we and others have implicated in the actin cytoskeleton organization8,12. A major defect in Strip1 mutant embryos is in the formation of the axial mesoderm (node and notochordal plate) and extension of the antero-posterior body axis. We have used SEM and WMIF to analyze the node and notochordal plate in wild-type (WT) and Strip1 mutant embryos as we show in the Representative Results and corresponding figures.
All experiments involving animal experiments were approved by the responsible authorities in North Rhein Westphalia (LANUV-NRW).
1. Scanning Electron Microscopy of the Mouse Embryonic Node
2. Whole Mount Immunofluorescence of the Mouse Node and Notochordal Plate
In order to examine the formation of the node in WT and Strip1 mutant embryos at ~ E7.5, we used SEM as described in Step 1 and shown in Figure 18. The ultrastructural details of the outside topology using SEM were quite informative and it was immediately clear that unlike the pit-shaped node in WT embryos, the mutant embryos had a flattened and irregular node. Higher magnification of the embryos showed the characteristic cilia on node cells that identified them unambiguously. The apparent lower density of cilia in the mutant might be attributable to the loss of node pit structure and curvature or a lower number of node cells. The notochordal plate which appears emanating from the node was also irregular in the mutant embryos. They were identifiable with their shorter cilia. Therefore, SEM was important to reveal the node morphogenesis defects in Strip1 mutants8. We have also used SEM in previous studies to show the absence of cilia in the embryonic node of mutants that lacked centrioles, which provide the template for cilia9.
To study the axial mesoderm formation defects in Strip1 mutant embryos at the cellular level, we used WMIF as described in Step 2 and shown in Figure 2. Using this technique, the node and notochordal plate were easily identified by F-Actin and T staining. WT node and notochordal plate cells have constricted apical domains where F-Actin was enriched, and nuclear T staining was evident. The notochordal plate extended rostrally in the WT but was short and irregular in the mutant. The data showed that F-Actin organization is abnormal in the different germ layers of the mutant embryos including the axial mesoderm8. Thus, WMIF was instrumental to study the defects in node and notochordal plate formation in Strip1 mutant embryos.
Figure 1. Scanning electron microscopy reveals the defects in node morphogenesis in Strip1 mutant mouse embryos. (Top) SEM analyses of WT and Strip1 mutant ventral embryonic nodes and notochordal plates (Noto)8. An example of a low magnification image of a WT embryo is shown on the left. (Bottom) Higher magnifications of the center of the nodes shown on top revealing the long monocilia projecting from the node cells. Anterior is up in all panels. Scale bars: 30 µm. Please click here to view a larger version of this figure.
Figure 2. Whole mount immunofluorescence shows the abnormal node and notochordal plate at the cellular level in Strip1 mutant embryos. (Top) Ventral 3D rendering (Volocity software) of WMIF on WT and Strip1 mutant embryos using a combination of fluorescence-conjugated phalloidin (F-Actin, red) and T antibody (green) staining. (Bottom) More examples of the staining shown above focusing on the node with higher zoom and including DAPI. Anterior is up in all panels. Scale bars: 30 µm. Please click here to view a larger version of this figure.
In this work, we demonstrate how to perform SEM and WMIF to visualize the mouse embryonic node and notochordal plate. The small size of gastrulating mouse embryos ~ E7.5 and the presence of these structures on the surface make them ideal to study using the techniques described2,7,8. The availability of good antibodies, such as T and cilia markers, gives excellent 3D information using WMIF on the structure, organization and formation of these essential embryonic organizers8.
Because mouse embryonic development proceeds at a very rapid pace and the node and notochordal plate are only transiently present on the surface of the embryo, timing is essential for the success of these experiments2,3. For example, 2-4 somite embryos are good for SEM analysis of a mature node pit with long cilia. In much earlier or later embryos (for example, 12 h before or after), the node might not be present on the surface. WMIF is a little more flexible in this regard but the structures themselves are also transient during development and the timing in this case depends on the researchers' interests.
The purity of the reagents is also essential for the success of these techniques, especially in probing the ultrastructure by SEM. Tiny impurities that stick to the embryos usually result in huge artifacts.
We have tested two different methods of embryo fixation for SEM one using half Karnovsky's fixative (2.5% glutaraldehyde, 2% paraformaldehyde and 0.1 M cacodylate buffer) and a simpler 2.5% glutaraldehyde in 1x PBS. We prefer to use the glutaraldehyde and PBS fixative as described in Step 1, however, we and others have also used the half Karnovsky's fixative successfully for SEM.
We have also compared two methods of drying the embryos for SEM and found no difference in the quality of the sample by using either a critical point dryer or HMDS as described in Step 1 and reported elsewhere14.
For Step 2, we tested embedding the embryos after the final washing steps in 1% low melting agarose mounted on a 35 mm glass-bottom dish and then topping it with ~ 10 µL of mounting medium. This embedding method works and preserves the original 3D structure of the embryo and associated structures; however, a multiphoton microscope is required to image the specimen because a regular confocal microscope cannot reach as deep into the intact embryos (~ 1 mm).
We believe that using these two techniques gives complementary information on the structure of the node and the notochordal plate during normal development and in mutants which show defects in the formation of these structures.
The authors have nothing to disclose.
H.B. is supported by startup funding from the Medical Faculty and SFB829 of the University of Cologne. C.X. is supported by DFG grant BA 5810/1-1. We would like to thank the Imaging Facilities at the CECAD research center and Memorial Sloan Kettering Cancer Center (New York, USA). We thank Joaquín Grego-Bessa (Spanish National Center for Cardiovascular Research, Madrid, Spain) for his insight on mounting the embryos for WMIF.
|1,1,1,3,3,3 Hexamethyldisilazane (HMDS)
|Critical Point Dryer
|Glutardialdehyde solution 25%
|SEM coating unit PS3
|Agar Aids for Electron Microscopy
|SEM microscope Quantum FEG 250
|ThermoFisher Scientific (FEI)
|Quantum FEG 250
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